Genera of soil bacteria that are members of the family Rhizobiaceae, are capable of infecting plants and inducing highly differentiated structures, root nodules, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant, most often of the family Leguminose, utilizes the ammonia as a source of nitrogen. Nodulating bacteria are classified in two taxonomically distinct groups, the fast-growing Rhizobium species and the slow-growing Bradyrhizobium species (Jordan, D. C. (1982) Int. J. Syst. Bacteriol. 32:136). Bradyrhizobium species include the commercially important soybean nodulating strains B. japonicum (i.e., strains USDA 110 and 123), promiscuous rhizobia of the cowpea group, and B. parasponia (formerly Parasponia Rhizobium) which nodulates the non-legume Parasponia, as well as a number of tropical legumes including cowpea and siratro. Bradyrhizobium japonicum strains have a narrow host range for nodulation generally limited to soybean, Glycine max. Fast-growing Rhizobium include, among others, Rhizobium trifolii, R. meliloti and R. leguminosarum, which nodulate clover, alfalfa and pea, respectively. These Rhizobium species also generally display narrow plant host range for nodulation. Rhizobium fredii, formerly R. japonicum, have a broader host range for nodulation including Glycine max cv. Peking, but not commercially useful soybean cultivars.
Nodulation and the development of effective symbiosis is a complex process requiring both bacterial and plant genes. Several recent reviews of the genetics of the Rhizobium-legume interaction are found in Broughton, W. J., ed. (1982) Nitrogen Fixation, Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. ed. (1983) Molecular Genetics of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay, A. A. and Leglocki, R. P., eds. (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction (Cornell University Publishers, Ithaca, N.Y.); Long. S. R. (1984) in Plant Microbe Interactions Volume 1, Kosuge, T. and Nester, E. W., eds. (MacMillan, New York) pp. 265-306; and Verma, D. P. S. and Long, S. L. (1983) International Review of Cytology (Suppl. 14), Jeon, K. W. (ed.), (Academic Press, N.Y.) p. 211-245.
In Rhizobium species genes required for nodulation and nitrogen fixation are located on large Sym plasmids. Although the process of recognition, infection and nodule development is complex, it appears that at least for the fast-growing rhizobia relatively few bacterial genes are directly involved, and these are closely linked on the Sym plasmid. See Schofield et al. (1984) Plant Mol. Biol. 3:3-11; (Downie et al (1983) Mol. Gen. Genet. 190:350-365; and Kodorosi et al. (1984) Mol. Gen. Genet. 193:445-452). In contrast, no Sym plasmids have been associated with B. japonicum strains. The nitrogenase and nodulation genes of these organisms are believed to be encoded on the bacterial chromosome.
Nodulation genes are those genes associated with non-nodulation and delayed nodulation phenotypes. Several nodulation genes designated nodABC and D, which are functionally and structurally conserved among the fast-growing Rhizobium, have been identified by hybridization studies and cross-species complementation experiments. These genes are designated the common nod genes. Recently it has been reported that B. parasponia (Marvel et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:5841-5845) and B. japonicum (Russel et al. (1985) J. Bacteriol. 164:1301-1308) contain nodulation genes which can functionally complement mutations in fast-growing Rhizobium and which show considerable structural homology to nodulation gene regions of R. meliloti and R. leguminosarum. Structural conservation between Rhizobium and Bradyrhizobium extends to other nodulation genes, nodI, J. Stacy et al. (1987) in Molecular Genetics of Plant-Microbe Interactions, Verma and Brisson (eds.) Martinus Nijhoff Publishers, Dordrecht, Netherlands, pp. 197-201, have recently reviewed the genetics of nodulation in B. japonicum.
In Rhizobium strains the common nod region, nodA, B and C genes are grouped sequentially and are coordinately transcribed as a single transcriptional unit. In some stranis, nodI and J are also reported to be part of this operon. The nodD gene is transcribed divergently from the nodABC(IJ) operon (Egelhoff et al. (1985) DNA 4:241-248; Jacobs et al. (1985) J. Bacteriol. 162:469-476; Rosen et al. (1984) Nucl. Acids Res. 12:9497-9508; and Torok et al. (1984) Nucl. Acids Res. 12:9509-9524). Divergent promoters for the nodABC operon and nodD are presumed to be located in the region between nodD and nodA.
Some Rhizobium strains contain more than one nodD-like gene. The presence of multiple nodD-like genes was first reported in R. fredii USDA 191 ((Appelbaum et al. (1985) in Nitrogen Fixation Research Progress, Marginus Nijhoff Publishers, Dordrecht, Netherlands, pp. 101-107) and subsequently in R. meliloti and other rhizobia (Gottfert et al. (1986) J. Mol. Biol. 191:411-420). Only one nodD gene has been reported in R. trifolii and R. leguminosarum. The presence of multiple nodD-like genes has not been reported in B. japonicum.
The functions of the multiple nodD like genes in the nodulation process or its regulation are not yet understood.
Regulation of the nodulation genes in Rhizobium species has been investigated. Only one nodulation gene, nodD, is reported to be constitutively expressed. Expression of the other nodulation genes including nodABCEFGHI and J requires the presence of appropriate exudate and a functional of nodD (Mulligan and Long (1985) Proc. Natl. Acad. Sci. U.S.A. 82:6609-6613; Rossen et al. (1985) EMBO J. 4:3369-3373; Innes et al. (1985) Mol. Gen. Genet. 201:426-432. Conserved DNA sequence elements are found within the promoter regions of the nodABC(IJ) operon in R trifolii, R. meliloti, R. leguminosarum, and in the nodEF operon and nodH in R trifolii. (Scott et al. (1985); Rolfe et al. (1985); Kondorosi et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds) Martinus Nighoff, Dordrecth, The Netherlands p. 73-75). These consensus sequences are associated with exudate-inducible expression of the nod genes and their presence in various Rhizobium suggest a conserved regulatory mechanism.
The presence of nod-box conserved sequences in nodulation promoter regions in B. parasponia (Scott (1986) Nucleic Acids Res. 14:2905-2919) and B. japonicum (Appelbaum et al. (1986) U.S. patent application Ser. No. 875,297, filed Jun. 17, 1986; and Stacy et al., 1987) has been reported. The presence of these regulatory sequences strongly suggests conservation of nodulation gene regulation in Bradyrhizobium and Rhizobium.
Nodulation genes of Bradyrhizobium strains as their analogues in Rhizobium strains affect the early stages of nodule formation including host-bacterium recognition, infection and nodule development. Wild type strains of Bradyrhizobium species display some variation in these early nodulation steps which is reflected in differences in relative rates of initiation of nodulation and ultimately in differences in competitiveness between strains for nodula occupancy. For example, B. japonicum USDA 123 is believed to be more competitive for nodulation than B. japonicum USDA 110. Strains which initiate infection and nodules earlier will occupy a greater portion of the nodules on a given plant. Improving the competitiveness of a specific Bradyrhizobium is an important part of the development of improved inocula for legumes. A more effective Bradyrhizobium strain, which would likely constitute an improved inoculum, must be able to out-compete the indigenous rhizobia population for nodule occupancy in order for their improved qualities to impact on the inoculated legume. An inoculating composition and/or an inoculating method which would improve competitiveness of a selected inoculant strain is therefore of significant commercial importance.
The establishment of nitrogen-fixing nodules is a multistage process involving coordinated morphological changes in both bacterium and plant requiring precise gene regulation. It has been suggested that an exchange of signals between plant and bacterium is requisite for mutual recognition and coordination of the steps of infection and nodulation development (Nutman, P. S. (1965) in Ecology of Soil Borne Pathogens, eds. F. K. Baker and W. C. Snyder, University of California Press, Berkeley, pp. 231-147; Bauer, W. D. (1981) Ann. Rev. Plant Phys. 32:407-449; and Schmidt, E. E. (1979) Ann. Rev. Microbiol. 33:355-376). The regulation of nodulation genes of rhizobia by chemical factors excreted by legumes in exudates is a specific demonstration of communication between host and symbiont.
Legume exudates have been previously linked to both simulation (Thornton (1929) Proc. Royal Soc. B 164:481; Valera and Alexander (1965) J. Bacteriol. 89:1134-1139; Peters and Alexander (1966) Soil Science 102:380-387) and inhibition (Turner (1955) Annals Botany 19:149-160; and Nutman (1953) Annals Botany 17:95-126) of nodulation by rhizobia.
Turner (1955) reported that addition of activated charcoal to rooting medium of clover plants led to an increased rate of nodule initiation. Activated charcoal was demonstrated to remove by adsorption an unidentified inhibitory substance secreted by clover roots. Nutman (1953) reported that clover roots excreted a substance inhibitory to nodulation. The substance was not identified but was found to be associated with the stage of nodulation on the plant. In both cases, it was suggested that both stimulatory and inhibitory factors were present in the root exudate.
Valera and Alexander (1965) and Peters and Alexander (1966) reported a nodulation enhancing factor in legume exudates that was dialyzable, water soluble and thermostable. This factor was replaceable by coconut water. More recently, Baghwat and Thomas (1982) Applied Environ. Microbiol. 43:800-805 described a stimulatory factor from legume exudates that was thermostable, was high molecular weight (about 2.times.10.sup.5) and was composed of protein and neutral hexoses. This factor was associated with elimination of nodulation delay in a certain cowpea Rhizobium strain. Halverson and Stacey (1984) Plant Physiol. 74:84-89; and (1985) Plant Physiol. 77:621-624 reported an exudate factor having a similar affect on nodulation initiation in B. japonicum USDA 110 mutants. In contrast to Baghwat and Thomas (1982), this stimulator of nodulation was described as a heat and trypsin sensitive protein, a galactose-specific lectin.
In addition to factors present in legume exudate, diverse chemicals have been identified as stimulators or inhibitors of nodulation. Reported stimulators of nodulation include inositol, indole, 2-phenol-n-butyric acid, D-leucine, barbituric acid, pyridine-3-sulfonate and quercetin (Molina and Alexander (1967) Can. J. Microbiol. 13:819-827; and Weir (1960) Phyton 15:109-118).
The specific components of legume exudates that act to induce nodulation gene expression in several species of Rhizobium have recently been identified. In addition, a number of compounds related in structure to the inducer components of exudate have also been identified as inducers of Rhizobium nod genes.
Peters et al. (1986) Science 233:977-980 identified luteolin (3',4',5,7-tetrahydroxyflavone) as the component of alfalfa exudates that induces nodABC expression in R. Meliloti. Nod gene induction was assayed as .beta.-galactosidase expressed from a lacZ gene which had been fused to the nodC gene of R. meliloti. In this gene fusion, the lacZ structural gene was placed under the regulatory control of the nodABC promoter and its associated nod-box regulatory sequence. A number of chemical compounds structurally related to luteolin were assayed for nod-gene induction in this system including several flavones, flavanones and flavanols. Of those compounds tested, only apigenin was found to induce the R. meliloti nod gene. Apigenin was found to be a much weaker inducer than luteolin.
Using similar nod-lacZ fusions in several nod genes, Redmond et al. (1986) Nature 323:632-635 reported the identification of three clover exudate components that induced expression of R. trifolii nod genes: 4',7-dihydroxyflavone (DHF), geraldone (3'-methoxy DHF) and 4'-hydroxy-7-methoxyflavone. In related work, Rolfe et al. (1986) U.S. patent application Ser. No. 844,870, filed Mar. 27, 1986, now abandoned, a number of substituted flavones, flavanols and flavanones were identified as R. trifolii nod gene inducers including luteolin and naringenin. Induction activity was reported to be confined to molecules having the flavone ring structure, in particular the isoflavones, daidzein and formononetin and coumestrol were inactive for R. trifolii nod gene induction.
Two of the nodulation gene inducers of R. leguminosarum from pea exudate were identified as eriodictyol (3',4', 5,7-tetrahydroxyflavanone) and apigenin-7-O-glucoside by Firmin et al. (1986) Nature 324:90-92. Apigenin, hesperitin and naringenin, in addition to other flavones and flavanones were also found to be active as inducers. The isoflavones daidzein, genistein and kaempferol were reported to be antagonists which strongly inhibited the activation of nod genes by inducers.
Zaat et al. (1987) J. Bacteriol. 169:198-204 characterized a R. leguminosarum nodulation gene inducer from Vicia sativa exudate as "flavonoid in nature, most likely a flavanone." Although the exudate component was not identified, naringenin, eridodictyol, apigenin and luteolin were reported to be strong nod inducers; 7-hydroxyflavone, a somewhat weaker inducer, and chrysin and kaempferol were weak or poor inducers. Among others, the isoflavones daidzein, genistein and prunetin were reported to be inactive.
The present work reports the identification of nodulation gene inducer compounds which activate expression of nodulation genes of Bradyrhizobium japonicum. These inducer compounds are useful in general for selective induction of genes placed under the regulatory control of B. japonicum soybean exudate inducible promoters and as components of soybean inoculation compositions.